Proteases belong to the industrially most important enzymes in general. Among these, in turn, serine proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62) which contain catalytically active amino acids are particularly important. These enzymes are nonspecific endopeptidases, i.e. they hydrolyze acid amide bonds which lie in the interior of peptides or proteins. Their pH optimum usually lies in the distinctly alkaline range. The article “Subtilases: Subtilisin-like Proteases” by R. Siezen, pages 75-95 in “Subtilisin enzymes”, edited by R. Bott and C. Betzel, New York, 1996, for example, offers an overview of this family. Subtilases are naturally formed from microorganisms. Among these subtilases, the subtilisins formed and secreted by Bacillus species are of particular interest.
Proteases are established, active ingredients present in a variety of detergents and cleaners as they catalyze the breakdown of protein-containing soil on the goods to be cleaned. Ideally, synergistic effects result between the enzymes and the other constituents present in the compositions concerned. Among the detergent and cleaner proteases, subtilases are particularly preferred due to their favorable enzymatic properties, including stability and pH optimum. In addition, they are also suitable for a large number of further industrial uses, for example as constituents of cosmetics and in the synthesis of organic chemicals.
Microorganism-containing samples from natural habitats may be cultured under conditions suitable for the production of novel enzymes, e.g., under alkaline conditions. In this way, novel alkaline proteases may be isolated. The microorganisms producing the most efficient enzymes are then selected for and purified, for example by means of plating out on protein-containing agar plates and measuring the lysis halos formed. Optionally, the genes encoding the proteases may be cloned. Such a procedure is described, for example, in the textbook “Alkalophilic Mikroorganisms. A new microbial world” by K. Horikoshi and T. Akiba (1982), Japan Scientific Societies Press, Springer-Verlag, N.Y., Heidelberg, Berlin, ISBN 0-387-10924-2, chapter 2, pages 9-26.
Notably, microbially-produced alkaline proteases are already employed in detergents and cleaners. For example, see WO 93/07276 A1 which describes the protease 164-A1 from Chemgen Corp., Gaithersburg, Md., USA, and Vista Chemical Company, Austin, Tex., USA, obtainable from Bacillus spec. 164-A1 is suitable for use in detergents and cleaners. Other examples are the alkaline protease from Bacillus sp. PD138, NCIMB 40338 from Novozymes A/S, Bagsvaerd, Denmark, (WO 93/18140 A1), the proteinase K-16 from Kao Corp., Tokyo, Japan, (U.S. Pat. No. 5,344,770) originating from Bacillus sp. ferm. BP-3376 and the protease described in WO 96/25489 A1 (Procter & Gamble, Cincinatti, Ohio, USA) from the psychrophilic organism Flavobacterium balustinum. 
Natural proteases may be optimized, for use in detergents and cleaners, via mutagenesis methods known in the art. Such methods include point mutagenesis, (e.g., generation of deletion, or insertion mutants) or fusion with other proteins or protein parts. The strategy of introducing specific point mutations into the known subtilisin molecules, in order to improve the washing performance, is also referred to as rational protein design. A similar strategy to improve performance entails modifying the surface charges and/or the isoelectric point of the molecules in order to modulate their interactions with the substrate via the introduction of point mutations. A further, supplementary strategy consists of increasing the stability of the proteases thereby increasing their efficacy. Stabilization by means of coupling to a polymer is described for proteases used in cosmetics, for example in U.S. Pat. No. 5,230,891. Such proteases exhibit improved skin compatibility. However, for detergents and cleaners, stabilization by point mutations is more commonly employed.
A new approach in enzyme development entails combining elements of related, known proteins thereby generating novel enzymes having improved functional properties. Such methods are also referred to as “directed evolution.” These include, without limitation: The StEP method (Zhao et al. (1998), Nat. Biotechnol., volume 16, pp. 258-261), Random priming recombination (Shao et al., (1998), Nucleic Acids Res., volume 26, pp. 681-683), DNA shuffling (Stemmer, W.P.C. (1994), Nature, volume 370, pp. 389-391) or RACHITT (Coco, W. M. et al. (2001), Nat. Biotechnol., volume 19, pp. 354-359). A further shuffling method referred to as “Recombining ligation reaction” (RLR) is described in WO 00/09679 A1.
Below, an overview of the industrially most important alkaline proteases of the subtilisin type is provided. Subtilisin BPN′, which originates from Bacillus amylotiquefaciens, or B. subtilis, is described by Vasantha et al. (1984) in J. Bacteriol., volume 159, pp. 811-819 and of J. A. Wells et al. (1983) in Nucleic Acids Research, volume 11, pp. 7911-7925. Subtilisin BPN′ is used as a reference enzyme with respect to numbering of amino acid positions in the subtilisins.
For example, the position of point mutations in subtilisin described in Application EP 251446 A1, are indicated using the numbering of BPN′ as a reference. Procter & Gamble Corp., of Cincinnati, Ohio, USA, refer to this material as “Protease B.” The BPN′ variants of Application EP 199404 A1 are referred to by Procter & Gamble Corp. as “Protease A.” “A Protease C” is in turn characterized, according to Application WO 91/06637 A1, by further point mutations of BPN′. “Protease D” refers, according to WO 95/10591 A1, to variants of the protease from Bacillus lentus. 
The protease subtilisin Carlsberg is described in the publications of E. L. Smith et al. (1968) in J. Biol. Chem., Volume 243, pp. 2184-2191, and of Jacobs et al. (1985) in Nucl. Acids Res., Volume 13, pp. 8913-8926. It is formed naturally by Bacillus licheniformis, and was and is obtainable under the trade name Maxatase® from Genencor International Inc., Rochester, N.Y., USA, and under the trade name Alcalase® from Novozymes A/S, Bagsvaerd, Denmark.
Protease PB92 is produced naturally by the alkalophilic bacterium Bacillus nov. spec. 92 and is obtainable under the trade name Maxacal® from the Gist-Brocades company, Delft, Netherlands. It is described in its original sequence in Patent Application EP 283075 A2.
Subtilisins 147 and 309 are marketed under the trade names Esperase® and Savinase®, respectively, by Novozymes. They were originally obtained from Bacillus strains that are disclosed by Application GB 1243784 A.
Subtilisin DY was originally described by Nedkov et al. 1985 in Biol. Chem Hoppe-Seyler, Volume 366, pp. 421-430.
The alkaline protease from B. lentus is an alkaline protease from Bacillus species and is described in Application WO 91/02792 A1. It natively possesses comparatively good stability with respect to oxidation and the action of detergents. Application WO 91/02792 A1 and Patents EP 493398 B1 and U.S. Pat. No. 5,352,604 describe its heterologous expression in the host B. licheniformis ATCC 53926. The claims of the aforesaid US Patent refer to positions 208, 210, 212, 213, and 268 as characteristic of the B. lentus alkaline protease; they correspond, in the numbering of the mature protein, to positions 97, 99, 101, 102, and 157. However this enzyme differs from the mature subtilisin 309 (Savinase®). The three-dimensional structure of this enzyme is described in the publication of Goddette et al. (1992) in J. Mol. Biol., Volume 228, pp. 580-595: “The crystal structure of the Bacillus lentus alkaline protease, Subtilisin BL, at 1.4 Å resolution.” Industrially important variants of this enzyme that are stabilized by point mutagenesis and are suitable in particular for use in washing and cleaning products are disclosed, inter alia, in Applications WO 92/21760 A1, WO 95/23221 A1, WO 02/088340 A2, and WO 03/038082 A2.
The enzyme thermitase, formed naturally by Thermoactinomyces vulgaris, was originally described by Meloun et al. (FEBS Lett. 1983, pp. 195-200). This is a molecule that as a whole exhibits substantial sequence discrepancies compared with the other subtilisins. The homology between the mature thermitase and the alkaline protease proteins from B. lentus DSM 5483 (see below) is not very high, (e.g., 45% identity; 62% similar amino acids).
Proteinase K is also a protease that exhibits comparatively low homology with the alkaline protease from B. lentus: only 33% identity (46% similar amino acids) at the level of the mature proteins. Proteinase K derives originally from the microorganism Tritirachium album Limber, and has been described by K.-D. Jany and B. Mayer (1985) in Biol. Chem. Hoppe-Seyler, Vol. 366, pp. 485-492.
WO 88/07581 A1 discloses proteases TW3 and TW7, which are very similar to one another, for use inter alia in washing and cleaning products.
Bacillopeptidase F from Bacillus subtilis possesses only 30% identity to the B. lentus alkaline protease at the amino-acid level. This enzyme is discussed in the aforementioned work by Siezen et al., but has not hitherto been described or claimed for use in washing and cleaning products.
Application WO 01/68821 A2 describes new subtilisins having good performance with respect to egg stains.
Further alkaline proteases that are formed from microorganisms that can be isolated from natural habitats are described in Applications WO 03/054185 A1 (from Bacillus gibsonii (DSM 14391)), WO 03/056017 A2 (from Bacillus sp. (DSM 14390)), WO 03/055974 A2 (from Bacillus sp. (DSM 14392)), and WO 03/054184 A1 (from Bacillus gibsonii (DSM 14393)). All these Applications also disclose corresponding washing and cleaning products containing these novel alkaline proteases.
A further group of industrially important proteases are the metalloproteases, e.g., enzymes that require a metal cation as a cofactor. Representatives of these are also assigned to the family of subtilases. For instance, metalloproteases from gram-positive microorganisms such as B. subtilis, but also from S. cerevisiae, S. pombe, E. coli and H. influenzae, are described in US 2003/0113895 A1. WO 00/60042 A1 and WO 02136727 A1, disclose detergents and cleaners containing metalloproteases. DE 10360805.2 discloses an alkaline metalloprotease whose encoding DNA is obtainable from a soil sample, and its use in detergents and cleaners.
A large number of novel proteases are described in WO 20041033668 A2. StmPr2 from Stenotrophomonas maltophilia, which is deposited under the entry AY253983 in GenBank (National Center for Biotechnology Information NCBI, National Institutes of Health, Bethesda, Md., USA) has also been previously described.
Further known protease enzymes are obtainable under the trade names Durazym®, Relase®, Everlase®, Nafizym, Natalase® and Kannase® from Novozymes, under the trade names Maxapem®, Purafect®, Purafect OxP® and Properase® from Genencor, under the trade name Protosol® from Advanced Biochemicals Ltd., Thane, India and under the trade name Wuxi® from Wuxi Snyder Bioproducts Ltd., China.
In light of the foregoing, it is clear that there is a great need for industrially employable proteases which exhibit altered activities from previously known proteases, particularly for their use in detergents and cleaners. A suitable protease for detergents or cleaners should exhibit a certain insensitivity to conditions suitable for cleaning—e.g., the presence of surfactants which are denaturing, of bleach, and high temperatures etc.—and also exhibit catalytic activity against appropriate substrates such as the proteins found in food residues.
There also exists a need for new alkaline proteases, which are naturally obtainable but which are also amenable to further optimization by means of various mutagenesis strategies. Such novel proteases may be generated using recently established shuffling technologies. Nucleotide sequences (even if the encoded enzyme exhibits comparatively modest performance) can be shuffled to produce new variants and thus in turn provide entirely new artificial enzymes for use in a variety of industrial applications.